Journal of Pharmacological and Toxicological Methods 71 (2015) 129–136

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Original article

Transmural dispersion of repolarization and cardiac remodeling in ventricles of rabbit with right ventricular hypertrophy Pradtana Meedech a, Nakkawee Saengklub b, Vudhiporn Limprasutr b, Sarinee Kalandakanond-Thongsong b,c, Anusak Kijtawornrat b,c,⁎, Robert L. Hamlin d a

Interdisciplinary Program of Physiology, Graduate School, Chulalongkorn University, Bangkok 10330, Thailand Department of Physiology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok 10330, Thailand Research study and testing of drug's effect related to cardiovascular system in laboratory animal research clusters, Chulalongkorn University, Bangkok 10330, Thailand d QTest Labs, LLC., 6456 Fiesta Dr., Columbus, OH 43235, United States b c

a r t i c l e

i n f o

Article history: Received 3 February 2014 Accepted 30 September 2014 Available online 12 October 2014 Keywords: Cardiac Dispersion Hypertrophy Rabbit Remodeling Repolarization Ventricle

a b s t r a c t Introduction: Recent publications demonstrated that rabbits with right ventricular hypertrophy (RVH) possess high sensitivity and specificity for drug-induced arrhythmias. However, the underlying mechanism has not been elucidated. This study aimed to evaluate RVH induced changes in cardiac remodeling especially the transmural dispersion of repolarization (TDR), epicardial monophasic action potentials (MAP), and hERG mRNA expression in rabbits. Methods: New Zealand White rabbits (n = 13) were divided into 2 groups: sham operated (SHAM, n = 6) and pulmonary artery banding (PAB, n = 7). PAB was induced by narrowing the pulmonary artery. Twenty weeks after surgery, hemodynamic, cardiac function, electrocardiograms, and MAP were obtained from PAB compared with SHAM. After measurement, rabbits were sacrificed to collect ventricular myocardium for histopathological analysis and measurement of hERG mRNA expression by real time PCR. Results: After 20 weeks, the % HW to BW ratio of whole heart and right ventricle (RV) and left and right ventricular free wall thickness was significantly increased in PAB when compared with those in SHAM. PAB has a significant electrical remodeling as demonstrated by lengthening of QT, QTc intervals, and increased Tp–Te duration. PAB also has a significant functional remodeling verified by decreased contractility index of RV and lengthened time constant of relaxation of LV. MAP of RV epicardium was significantly shortened in PAB consistently with increased hERG mRNA expression at the epicardium of RV. Discussion: The rabbit with PAB demonstrates cardiac remodeling diastolic and systolic dysfunctions. These rabbits also demonstrate increased TDR and electrical remodeling related to the change of hERG mRNA expression which may be prone to develop arrhythmias. © 2014 Elsevier Inc. All rights reserved.

1. Introduction Right ventricular hypertrophy (RVH) is an increase in size of the right ventricular muscle or an enlargement of the right ventricular chamber of the heart. This pathological condition may result from pulmonary artery stenosis (PS), tetralogy of Fallot, pulmonary artery hypertension (PAH), essential hypertension or pulmonary diseases (Haddad, Hunt, & Rosenthal, 2008). Several clinical studies demonstrated that RVH prevalence in systemic hypertension patients varied from 17 to 80% (Cuspidi, Sala, Muiesan, Luca, & Schillac, 2013). A consequence of an increase in ventricular mass is the development of diastolic and systolic dysfunctions, right heart failure, and sudden cardiac death (Gan et al., 2007; Piao et al., 2010). Recently, Humbert, Sitbon, and Chaouat (2010) reported that the overall mortality rate of patients ⁎ Corresponding author at: Department of Physiology, Faculty of Veterinary Science, Chulalongkorn University, Henri-Dunant Rd, Pathumwan, Bangkok 10330, Thailand. Tel.: +66 91 0097246. E-mail address: [email protected] (A. Kijtawornrat).

http://dx.doi.org/10.1016/j.vascn.2014.09.012 1056-8719/© 2014 Elsevier Inc. All rights reserved.

with RVH due to PAH was 30–50% in which 17–28% of that mortality rate was sudden cardiac death. The major cause of the sudden cardiac death may result from spontaneous ventricular fibrillation (Umar et al., 2012). This arrhythmia has been demonstrated to associate with electrical and structural remodeling of the right ventricle (Lee, Kodoma, Anno, Kamiya, & Toyama, 1997; Umar et al., 2012). In patients with RVH due to PAH, remodeling of right ventricle resulted in increased heterogeneity of ventricular action potential duration, a substrate for reentry arrhythmia (Henkens, Scherptong, & Kralingen, 2008). In animal model of ventricular hypertrophy, Panyasing, Kijtawornrat, Rio, Carnes, and Hamlin (2010) have shown that rabbits with RVH tended to develop long QT syndrome and torsades de pointes in response to delayed rectifier potassium channel blocker infusion when compared to left ventricular hypertrophy and biventricular hypertrophy. Furthermore, QTc interval dispersion was also seen in both humans and animals with RVH (Panyasing et al., 2010; Tuncer, Gunes, & Guntekin, 2008). However, to date a systemic study of cardiac remodeling (i.e., electrophysiology, hemodynamic, ventricular function, and anatomical study) of the right ventricular hypertrophy due to

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pulmonary artery stenosis and the role of human ether-a-go-go related gene (hERG) in the rabbit model of RVH is still lacking. The present study was designed to evaluate RVH induced changes in cardiac remodeling especially the transmural dispersion of repolarization, epicardial monophasic action potentials, and hERG mRNA expression in rabbits. 2. Methods 2.1. Animals Adult male New Zealand White rabbits, weighing between 2.3 and 2.9 kg, were used in this study. All animals were housed individually in a standard rabbit cage and all procedures were approved by the Faculty of Veterinary Science Animal Care and Use Committee, Chulalongkorn University. The rabbits were randomly divided into two groups: shamoperated group (SHAM, n = 6) or pulmonary artery banding group (PAB, n = 7). On the operating day, the rabbits were anesthetized with a combination of tiletamine hydrochloride and zolazepam hydrochloride (15 mg/kg, I.M.) and further maintained with 2–3% isoflurane via a customized facemask designed for small animals throughout the surgical procedures. Detail of surgical procedure was previously described by Panyasing et al. (2010). Briefly, the rabbits were placed in a dorsal recumbency position and a midline incision of approximately 1.5 in. in length was made after 2% lidocaine infiltration. The sternum was then cut and retracted so that the pericardial sac could be seen. The longitudinal cut was performed on the pericardium to reveal the pulmonary artery. The diameter of this vessel is approximately 6.0–6.4 mm for all rabbits used in this study. The pulmonary artery was constricted to approximately 3.2 mm at the origin of the vessel. Therefore, the vessel was about 50% narrowed. The pericardial sac was left open, the sternum, muscle layers, and subcutaneous were closed. All rabbits were given carprofen 4 mg/kg, I.M., SID for 3 days and 25 mg/kg enrofloxacin, I.M., SID for 7 days post-operatively. Rabbits in SHAM group were anesthetized and operated as in PAB group except for the banding of pulmonary artery. 2.2. Assessments of cardiac function and electrophysiology After 20 weeks, animals were anesthetized as previously described. The bipolar transthoracic electrocardiogram (ECG) was obtained. The high and low pass filters were set at 0.01 Hz and 1 kHz, respectively. Signals were digitally sampled at a frequency of 2 kHz. A cut-down technique was performed on the right internal carotid artery and the right jugular vein. For the right internal carotid artery, a 2 Fr micromanometer catheter (Millar instrument, Texas, USA) was inserted and placed at the level of aortic arch to measure aortic pressure (AoP). The left ventricular pressure (LVP) was recorded by advancing the catheter into the left ventricle. For the right jugular vein, another 2-Fr Millar catheter was inserted into the right atrium to obtain right atrial pressure (RAP). The right ventricular pressure (RVP) was recorded by advancing the catheter into the right ventricle. The monophasic action potential (MAP) was recorded for both right and left ventricular epicardia by placing MAP electrode catheter (EP technologies, Boston Scientific, MN, USA) directly on the epicardium for 1 min each following a midline incision over the sternum. The MAP was recorded for 1 min each. All data were recorded with an IOX program (IOX version 1.8.5, EMKA Technologies, Fall Church, VA, USA). 2.3. Measurement of heart weight and ventricular free wall thickness At the end of the experiment, all rabbits were euthanized with 200 mg/kg pentobarbital sodium while they were under general anesthesia. The heart was collected, rinsed with physiologic normal saline, patted dried and weighed. A cross-section of the left and right ventricles was made just below the coronary grove. Left and right ventricular free

wall thickness was measured at the level of the head of the papillary muscle. Weights of atria, and left and right ventricles were also collected. Samples of the left and right ventricular endocardium and epicardium were collected from the strip of myocardium sectioned from the free-wall at the level of the head of the papillary muscle. These muscle strips were immersed in RNAlater and kept at −80 °C for further analysis of mRNA expression. The rest of the tissue samples were collected in 10% formalin for histopathological analysis. The normalized heart weight was calculated using the following formula: ((whole heart weight (g) / body weight (kg)) ∗ 100) and presented as %HW/BW. 2.4. Data analysis The ECG was analyzed automatically by using ECG auto program (ECG auto version 3.3.0.15, EMKA Technologies, Falls Church, VA, USA). Electrocardiographic parameters included were RR, PQ, QRS, QT, and the duration from the peak of T wave to the end of T wave (Tp–Te). QT interval was measured from the beginning of Q wave to the end of T wave. The QT interval corrected for changes of heart rate, QTc interval, was calculated by using the Carlsson equation (Carlsson, Abrahamsson, Andersson, Duker, & Schiller-Linhardt, 1993). Tp–Te duration was measured from the peak of T wave to the end of T wave. MAPs of right and left ventricular epicardia were analyzed for action potential duration (APD) after it was repolarized for 90% (APD90). Recordings of AoP and RAP were analyzed for mean arterial pressure (MBP) and mean right atrial pressure, respectively. Recordings of left and right ventricular pressures were analyzed for indices of inotrope (contractility index, the dP/dtmax divided by the ventricular pressure at that point) and lusitrope (tau, ventricular relaxation time constant). Tau was calculated by Glantz method, P(t) = P0e− t⁄τE + Pα, where P = pressure at time t, P0 = amplitude constant, τE = Glantz relaxation constant, and Pα = non zero asymptote due to pleural and pericardial pressures (Raff & Glantz, 1981). 2.5. hERG mRNA expression analysis Total RNA was extracted from the myocardium of the left and right ventricles from all groups with Aurum™ Total RNA Fatty and Fibrous Kit (BioRad, Hercules, CA, USA) in accordance with the manufacturer's instruction. Subsequently, the total RNA (1 μg) was reverse-transcribed into complementary DNA (cDNA) using the iScript™ Reverse Transcription Supermix for RT-qPCR kit (Bio-Rad Laboratories Inc, Hercules, CA, USA). The synthesized cDNA was quantified with spectrophotometer and stored at −20 °C for later analysis. The real time PCR assays were performed with an ABI 7300 instrument (Applied Biosystem, Foster City, California, USA) using the Real-time-PCR Master Mix E4 (GeneOn, Ludwingshanfen am Rhein, Germany) in accordance with the manufacturer's protocol. For each PCR reaction, it composed of cDNA (1 μg), PCR master Mix, the forward and reverse primers (20 μM) and adjusted volume of nuclease-free water. All reactions were performed in duplicate. The PCR was performed under the following conditions: 95 °C for 3 min followed by 40 cycles of denaturation at 95 °C for 30 s, annealing and extension at 58 °C for 1 min. Fluorescent signals were detected at the end of the extension step of each cycle. A dissociation step, consisting of 94 °C for 3 min, 58 °C for 30 s and 72 °C for 1 min was performed at the end to confirm product specificity. The primer sequences used in this study were: hERG, forward primer, 5′-CAGGC ACCACGCATC CA-3′, reversed primer, 5′-GTCAGGGTGT GTCGGAACTT-3′;18 s rRNA, forward primer, 5′-CCG CGG TTC TAT TTT GTT GGT TTT-3′, reversed primer, 5′CGG GCC GGG TGA GGT TTC-3′. The accession numbers were as followed, hERG, OCU97513 and 18sRNA, AF102857. The quantification of hERG mRNA was calculated using the comparative threshold cycle method. The results are reported as relative gene expression. The 18S rRNA was used as an internal control, against which each target signal was normalized, this was referred as the ΔCt.

P. Meedech et al. / Journal of Pharmacological and Toxicological Methods 71 (2015) 129–136

The fold changes in mRNA expression were analyzed between groups and presented as fold change using the following formula: −ΔΔCt;

2

where : ΔΔCt ¼ ½ΔCtPAB group – avg:½ΔCTsham group :

The specificity of hERG and 18 s rRNA PCR products was confirmed with the dissociation curve following PCR and by using high resolution agarose gel electrophoresis. 2.6. Histomorphological study The heart tissues were fixed with 10% formaldehyde buffer for at least 48 h. A 3 μm serial section was performed for each ventricle and stained with hematoxylin and eosin (H&E stain), Masson trichrome stain, or Periodic acid-Schiff (PAS) stain. All sections from both groups of rabbits were evaluated by an experienced pathologist. 2.7. Statistical analysis All data were presented as mean ± SEM. Parameters of electrophysiology, hemodynamics, and cardiac function were compared between groups at 20 weeks after surgery by using student unpaired t-test. A probability value of p b 0.05 was considered to be significant. 3. Results In general, a total of 14 rabbits were anesthetized and underwent open chest surgery. One rabbit in pulmonary artery banding group died at the end of procedure due to pneumothorax. Therefore, the success rate of this procedure was 92.8% (SHAM, n = 6; PAB, n = 7). All survived rabbits did not show any sign of congestive heart failure (e.g., abdominal distension, lung congesting, dyspnea, anorexia, lethargy, etc.) during the study period (20 weeks). 3.1. Effects of PA banding on cardiac electrical remodeling The parameters of ECG and MAP were compared between SHAM and PAB groups after 20 weeks of surgery (Table 1 and Fig. 1). In response to chronic pressure overload produced by narrowing of the pulmonary artery, QT, QTc intervals and Tp-Te were significantly lengthened when compared to SHAM rabbits (p b 0.05, p b 0.01, and p b 0.01, respectively). The percent changes of QT, QTc, and Tp–Te in PAB from SHAM were 19.84, 15.80, and 123%, respectively. Chronic pressure overload did not alter RR, PQ, or QRS intervals. While the APD90 of the left ventricular epicardium did not change, the APD90 of the right ventricular epicardium was significantly shortened in PAB rabbits when compared to sham rabbits (p b 0.05). Table 1 Electrical remodeling of ventricle in SHAM operated rabbits (SHAM) and pulmonary artery banding (PAB) rabbits. Values were presented as mean ± SEM. Each point was calculated from a 1 minute data recording from anesthetized rabbits. *p b 0.05 and **p b 0.01 compared with SHAM by using student unpaired t-test. Parameters

SHAM (n = 6)

PAB (n = 7)

RR (ms) PQ (ms) QRS (ms) QT (ms) QTc (ms) Tp-Te (ms) MAPD90RV (ms) MAPD90LV (ms)

206.9 63.7 49.9 131.8 148.1 16.3 122.70 118.07

222.4 65.4 54.5 158.0 171.5 36.4 103.56 124.69

± ± ± ± ± ± ± ±

12.1 2.3 2.3 4.7 3.5 3.4 3.18 2.04

± ± ± ± ± ± ± ±

11.1 3.3 1.6 5.7* 4.5 ** 3.1** 4.05* 3.52

QTc = corrected QT interval for heart rate by Carlsson formula; Tp–Te = duration between the peak of T wave and the end of T wave; MAP90 = monophasic action potential duration at 90% of repolarization, RV = right ventricle; LV = left ventricle; ms = milliseconds.

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3.2. Effects of PA banding on cardiac functional remodeling The MBP in PAB group when compared to SHAM was increased significantly to 15.0% (p b 0.05) whereas the mean RAP in PAB rabbits tended to increase when compared to SHAM (Table 2). End diastolic pressure (EDP) of both RV and LV was 3.5 times higher in PAB than those in SHAM. End systolic pressure (ESP) of RV and LV was 1.7 and 1.1 times significantly higher in PAB than those in SHAM, respectively. Contractility index of right ventricle was significantly decreased (−44.68%) in PAB rabbits when compared to SHAM rabbits (p b 0.05) whereas it did not change for the left ventricle. The Tau of left ventricle was significantly lengthened (50.38%) for PAB rabbits (p b 0.05) when compared to SHAM while the Tau of the right ventricle for PAB rabbits tended to increase (32.33%, p = 0.1). 3.3. Effect of PA banding on cardiac anatomical remodeling Structural remodeling was determined by assessment of the heart weight/body weight ratio, ventricular free wall thickness, and morphological changes (Table 3). The %HW/BW in PAB rabbits was increased significantly (p b 0.05) for whole heart (13.0%) and for right ventricle (73.6%) when compared to SHAM rabbits. The left and right ventricular free wall thickness in PAB were increased significantly (p b 0.01) 30.49% and 49.32%, respectively when compared with SHAM. The cardiac myocytes of right ventricle appeared to be similar in size in both groups as observed with H&E stains. However, a Masson's trichrome stain showed marked fibroblast proliferation (blue color with black arrow, Fig. 2A–B) in PAB rabbits compared with SHAM rabbits. Moreover, a Periodic acid-Schiff (PAS) stain demonstrated a marked positive (glycogenmagenta in color with black arrow, Fig. 2C–D) in rabbits with PAB compared with SHAM rabbits. 3.4. The changes of hERG potassium channel mRNA expression in myocardium The fold changes of mRNA abundance of hERG to 18 s rRNA in left ventricular endocardium, left ventricular epicardium, right ventricular endocardium and right ventricular epicardium of SHAM and PAB rabbits were calculated. For the left ventricle, the expression of hERG potassium channel at the endocardium of the PAB rabbits was higher than the SHAM rabbits by 1.75 fold; while, there was no change observed in the epicardium. For the right ventricle, the expressions of hERG potassium channel in the endocardium and the epicardium of the PAB rabbits were higher than the SHAM rabbits by 1.32 and 1.53 fold, respectively. The specificity of each primer used in this study was confirmed by performing a high resolution gel electrophoresis and a dissociation curve at the end of PCR. A single band and a single peak were evidenced in an agarose gel electrophoresis and dissociation curves indicating primer specificity (data not shown). 4. Discussion and conclusion The present study was designed to test the hypotheses that 1) rabbits with RVH produced by narrowing the pulmonary artery may develop electrical, functional, and anatomical remodeling of the right ventricle and 2) hERG potassium channel mRNA expression of rabbits with RVH may be altered. Based on the results of this study, both hypotheses were accepted. 4.1. Electrical remodeling of the ventricle In the present study, PAB induced RVH and causes repolarization remodeling (increased QT and QTc intervals, decreased APD90 and elevated hERG mRNA expression of the RV epicardium) and increases transmural dispersion of repolarization (increased Tp–Te duration). In our previous study, torsades de pointes arrhythmias occurred

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Fig. 1. A representative electrocardiogram (ECG), left ventricular pressure (LVP), right ventricular pressure (RVP), and monophasic action potential (MAP) of the left ventricle (LV) and right ventricle (RV) of one rabbit with SHAM operated (SHAM) and one rabbit with pulmonary artery banding (PAB). The tracings were obtained at 20 weeks after surgery. Notice the deep S waves, J-point elevation, and increased amplitude of T waves following production of PAB and consistent with right ventricular enlargement.

predominantly in RVH rabbits (Panyasing et al., 2010). It has been demonstrated previously that electrical remodeling especially in the right ventricle is the proximate cause of arrhythmias (Benoist, Stones, Drinkhill, Bernus, & White, 2011; Tanaka, Takase, & Yao, 2013; Umar et al., 2012). In the current study, both prolongation of repolarization and increased transmural dispersion of repolarization were observed in rabbits with RVH suggesting the tendency to increase arrhythmogenic risk in PAB rabbits. Transmural dispersion of repolarization (TDR) is the difference between the longest and shortest repolarization of transmembrane action potentials. It has been shown that TDR is related to Tp–Te, time between the peak of T wave to the end of T wave (Antzelevitch, 2007; Roche, Kijtawornrat, Hamlin, & Hamlin, 2005). Recently, increased Tp–Te has been demonstrated to be associated with increased incidence of ventricular arrhythmias in Brugada syndrome and cardiac resynchronization therapy patients (Barbhaiya, Po, & Hanon, 2013; Letsas, Weber, Astheimer, Kalusche, & Arentz, 2010). The present study also demonstrated that epicardial action potential duration of right ventricle in rabbits with RVH was shortened when

compared with SHAM rabbits. However, the epicardial action potential duration of the left ventricle did not change. This result was supported by the higher expression of hERG potassium channel mRNA expression in the right ventricular epicardium. The expression of the IKr channel in the left ventricular epicardium in PAB was not changed when compared to SHAM. The hERG mRNA expression was higher in the endocardium of both left and right ventricles of rabbits with RVH than those of SHAM rabbits. These results yield both agree and disagree with previous studies (Han, Chartier, Li, & Nattel, 2001; Hu, Yan, Lin, Liu, & Li, 2011; Li, Lau, Ducharme, Tardif, & Nattel, 2002; Tsuji et al., 2000). The findings of IKr in disease heart are variable which could be due to the difference in species, the different zones of the heart (i.e., apex vs base, epicardium vs midmyocardium vs endocardium), and types of heart disease (Li et al., 2002; Panyasing et al., 2010; Tsuji, Zicha, Qi, Kodama, & Nattel, 2006; Volders et al., 1999). Some studies reported that the IKr was decreased in rabbit ventricle (Tsuji et al., 2000). Other studies demonstrated that IKr was not changed in canine heart failure (Han et al., 2001; Li et al., 2002). In rats with left ventricular hypertrophy, the down-regulation

P. Meedech et al. / Journal of Pharmacological and Toxicological Methods 71 (2015) 129–136 Table 2 Hemodynamic and cardiac function in sham operated rabbits (SHAM) and pulmonary artery banding (PAB) rabbits. Values were presented as mean ± SEM. Each point was calculated from a 1 minute data recording from anesthetized rabbits. *p b 0.05 and **p b 0.01 compared with SHAM by using student unpaired t-test. Parameters

SHAM (n = 6)

PAB (n = 7)

MBP (mm Hg) RAP (mm Hg) EDPRV (mm Hg) EDPLV (mm Hg) ESPRV (mm Hg) ESPLV (mm Hg) CIRV CILV TauRV (ms) TauLV (ms)

56.0 3.2 2.2 3.3 20.9 61.6 74.1 81.7 18.1 13.4

65.9 7.0 7.7 7.0 34.8 70.9 44.2 86.2 22.1 20.0

± ± ± ± ± ± ± ± ± ±

3.3 1.3 1.6 1.5 2.0 3.6 15.5 8.8 4.2 0.7

± ± ± ± ± ± ± ± ± ±

2.3* 1.8 1.2* 1.5 0.8** 1.7* 3.8* 6.8 1.8 2.6*

MBP = mean arterial blood pressure; RAP = mean right atrial pressure; EDP = end diastolic pressure; ESP = end systolic pressure; CI = contractility index (the maximal rate of rise of the ventricular pressure divided by the ventricular pressure at that point); Tau = the relaxation time constant; RV = right ventricle; LV = left ventricle; mm Hg = millimeter of mercury; ms = milliseconds.

of hERG mRNA expression was reported (Hu et al., 2011). Previous study has compared the expression of hERG and KvLQT1 (an IKs channel encoded by KCNQ1 gene) mRNA expression between tachypaced rabbits and bradypaced rabbits and found that hERG mRNA was downregulated only in bradypaced rabbits whereas KvLQT1 mRNA was downregulated in both groups (Tsuji et al., 2006). The lengthening of the QT and QTc intervals was observed in anesthetized PAB rabbits when compared with SHAM. However, shortening of the monophasic action potential was noticed at the right ventricular epicardium (APD90). On the other hand, the monophasic action potential at the left ventricular epicardium was not changed. The body surface (peripheral) ECG is the voltage difference between the first region of the heart activated and the last region activated. This is, in general, the voltage difference between the subendocardium at the apex (the first) and subepicardium at the base (the last) (Williams, 1982). In contrast, the MAP is the measurement of electrical activity of cells beneath the MAP electrode (diameter size = 0.2 mm), it does not represent the electrical activity of the whole heart (Franz, 1999). Thus, the QT is almost always longer at the body surface than at any point on the heart. Also, since the QTc contains RR interval in the calculations and RR may change, the QTc may not even be related with QT interval. This finding is in accordance with previous report by Danik et al. (2002). They investigated the ventricular MAP in open-chest mice and correlated repolarization with the ECG. Interestingly, they found no significant correlation by linear regression between APD90 and the ECG intervals. Shortening of the MAP (and effective refractory period) of the ventricle is known to predispose to arrhythmia mediated by reentry. Thus this model could prove useful to evaluate drug-induced predilection to arrhythmias mediated by shortening the effective refractory period. However, as shown in our previous study, QT became less stable (i.e., greater beat-to-beat variability) in rabbit with RVH, a finding known to be a better predictor of predilection to torsades de pointes than changing

Table 3 Anatomical remodeling of ventricle in sham operated rabbits (SHAM) and pulmonary artery banding (PAB) rabbits. Values were presented as mean ± SEM. Each point was calculated from 1 min data recording from anesthetized rabbits. *p b 0.05 and **p b 0.01 compared with SHAM by using student unpaired t-test. Parameters

SHAM (n = 6)

PAB (n = 7)

%HW/BW(WH) %HW/BW(RV) LVFWD (mm) RVFWD (mm)

0.24 0.031 4.10 2.21

0.27 0.054 5.35 3.30

± ± ± ±

0.01 0.002 0.28 0.31

± ± ± ±

0.01* 0.006* 0.25** 0.24**

HW = heart weight; BW = body weight; WH = whole heart; RV = right ventricle; LVFWD = left ventricular free wall diameter; RVFWD = right ventricular free wall diameter; mm = millimeters.

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mean QT or QTc. In the present study in the rabbit with RVH, MAPs were shortened in epicardium of the right ventricle whereas it tended to prolong in the epicardium of the left ventricle. This difference in duration reflects increased heterogeneity of repolarization, another electrophysiological substrate predisposing to arrhythmia. Finally it has been suggested that changes in cardiac wavelength λ [λ = effective refractory period (ERP) × conduction velocity (CV)] promote reentrant arrhythmias (Aidonidis et al., 2009). Since changes in APDs reflect the changes in ERP, therefore, agents that prolong APDs tend to increase the risk for torsades de pointes while agents that shorten APDs tend to increase the risk for ventricular fibrillation (Hondeghem, 2008; Hondeghem, Dujardin, Hoffmann, Dumotier, & De clerk, 2011). 4.2. Functional remodeling of the ventricle At 20 weeks after surgery, the contraction of the right ventricle was significantly decreased as suggested by contractility index. Recent studies in anesthetized guinea pigs and dogs demonstrated that contractility index is more reliable for assessment of inotropic state of the heart than dP/dtmax (Kijtawornrat et al., 2014). The relaxation of the right and left ventricle of rabbit with RVH also decreased significantly as assessed by tau (for only the left ventricle). The decrease in both myocardial contraction and relaxation after pulmonary banding suggested that the RVH rabbits possess systolic and diastolic dysfunctions. These results are in accordance with previous study in infant rabbits in which the cardiac function impaired beginning at 3 weeks after surgery (Minegishi, Kitahori, Murakami, & Ono, 2011). The current study showed that both ESP and MBP of the left ventricle increased in PAB rabbits. This is an expected finding with increased hindrance to RV ejection imposed by the PA banding. The fact that the contractility index for the RV decreased indicates that RV contractility decreased. That RVP increased despite a decrease in RV contractility, can be explained by increased RV preload and/or increased hindrance to ejection. Banding of the PA clearly increased hindrance to RV ejection. Therefore from this study we are unable to identify the precise mechanism for the increase in peak of right ventricular systolic pressure. The lesser but significant increase in ESPLV may be due to increase in contractility and/or preload and to increased hindrance to ejection imposed by the aorta (Louie et al., 1995). Recent studies suggested that there is a relationship between the two ventricles since they share the interventricular septum (Hill & Singal, 1997; Voelkel et al., 2006; Yu et al., 1996). Therefore, impairment of the right ventricular function may affect the function of the left ventricle (Schou, Peters, Kim, Frokiaer, & Nielsen, 2007). This might suggest that impaired RV function could translate to impaired LV function. In the rat model of right-sided heart failure produced by narrowing of the pulmonary artery, hypertrophy of the left ventricle and septum was observed as well as impairment of left ventricular function (Schou et al., 2007). However, LV function as assessed by peak systolic LVP increased. The findings describing LV function in this study may be explained by increased hindrance to LV ejection elevation identified as elevation of aortic pressure to 66 mm Hg from 56 mm Hg in the SHAM group. The increased aortic pressure may reflect unexpected stiffening of the systemic arterial tree in response to increased altered right ventricular physiology. 4.3. Anatomical remodeling of the ventricles Narrowing of pulmonary artery used in this study was adjusted to approximately 50% of the diameter of the pulmonary artery. This process gradually produces pressure-overload to the right ventricle as the rabbits grew. In the current study, the morphological remodeling was confirmed by a significant increase of %HW/BW ratio of the right ventricle and the whole heart as well as the thickening of right ventricular free wall. The histopathological results of right ventricle demonstrated a tendency for increase of fibrous tissue and glycogen replacement of the dead cardiac myocytes in PAB rabbit when compared with SHAM

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SHAM

PAB

A

B

C

D

Fig. 2. Histopathological section of right ventricular free wall stained with Masson's trichrome (A, B) and (C, D) in SHAM operated (SHAM) and pulmonary artery banding (PAB) rabbits. 40×; scale bar = 50 μM. Notice that the right ventricular myocytes appeared to be similar in size for both PAB and SHAM. In PAB group, the Masson's trichrome and Periodic acid-Schiff (PAS) stain demonstrated fibroblast proliferation (black arrow at blue color) and glycogen deposit (black arrow at magenta color), respectively.

rabbits. These results are consistent with previous studies in infant rabbits and adult rats which demonstrated that pulmonary artery banding induced pressure-overload, right ventricular hypertrophy, and apoptosis of the myocytes beginning at 4 weeks after banding and suggested that the apoptosis may lead to myocardial dysfunction (Braun, Szalai, Strasser, & Borst, 2003; Ikeda, Hamada, & Hiwada, 1999; Minegishi et al., 2011; Teiger et al., 1996). 4.4. Importance of the model and translational to humans It has been recognized that the incidence of cardiac arrhythmias increases in humans especially when the risks factors are existing (Roden & Yang, 2005). Previous studies demonstrated that right ventricular failure following pulmonary hypertension and right ventricular hypertrophy is associated with increased incidence of cardiac sudden death; however, the underlying mechanism is unclear (Umar et al., 2012). Therefore, there is a need for an animal model of right ventricular hypertrophy. Several methods have been used to induce right ventricular hypertrophy including monocrotaline-induced vasculitis, hypoxia, and PAB (Bonnet et al., 2004; Jones et al., 2004; Schou et al., 2007). Among these models, PAB is an encouraging method for producing RVH and right-sided HF. We believe that this PA banding model is superior to monocrotaline, since the banding is a single, non-chemical stimulus to impose stress on the RV, whereas the chemical, monocrotaline, no doubt imposes many other consequences in addition to vasculitis. In the rabbit, the surgical procedure is quick and simple, requiring b15 min per rabbit. It does not require elaborate instrumentation or positive pressure ventilation, since the pleural cavity is not broached. After banding, the right ventricle encounters chronic pressure overload and the adaptive responses, including remodeling of the ventricle (Voelkel et al., 2006) occur rapidly. However, the rabbit model of RVH

does not show the clinical symptoms of heart failure even after 20 weeks of 50% narrowing of the pulmonary trunk. Heart failure could be produced by greater narrowing of the pulmonary trunk and/or by production of tricuspid regurgitation. This model could be useful for elucidating the mechanism(s) by which pulmonary hypertension, ventricular remodeling, and arrhythmias occur. Moreover, the model has been shown to possess more sensitivity and specificity to assess drug-induced torsades de pointes than rabbits even with iatrogenic bi-ventricular hypertrophy or left ventricular hypertrophy (Panyasing et al., 2010). Future studies using this model and information gained from our previous study could provide pharmacologists and toxicologists insight into mechanism(s) of remodeling and arrhythmogensis. This model could be used for studies in safety pharmacology evaluating a wide spectrum of potential therapeutic modalities. In humans, it is known that pulmonary hypertension and right ventricular failure are associated with increased incidence of cardiac sudden death (Zipes & Wellens, 1998). Cardiac arrhythmias have been suggested as a possible mechanism (Umar et al., 2012). To the extent of our finding in the rabbit model of RVH, cardiac remodeling (functional, anatomical, and electrical) including increased TDR and heterogeneity of hERG mRNA expression in the ventricles may be considered a potential mechanism of arrhythmias in humans with RVH. 4.5. Conclusions Overall results of this study indicated that rabbits with PAB for 20 weeks developed right ventricular hypertrophy as suggested by electrical, functional, and anatomical remodeling. The electrical remodeling included prolongation of repolarization, increased transmural dispersion of repolarization, and shortening of APD90 of right ventricular

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epicardium which was related to overexpression of hERG mRNA potassium channel. The functional remodeling included impaired right and left ventricular functions. The anatomical remodeling included thickening of the left and right ventricular free walls, increased %HW/BW ratio, marked positive PAS stain, and marked fibroblast proliferation. 4.6. Future studies Future studies need to assess the TDR as a biomarker for drug-induced torsades de pointes in the model of RVH by administration of both known torsadogenic and non-torsadogenic compounds to the rabbits. In addition, a gold standard for assessment of cardiac function, a pressure– volume loop relationship, should be used in the future experiment. Moreover, the apoptotic biomarkers (i.e., DNA fragmentation assessed by TUNEL assay) and cardiac signaling molecules would be utilized to check the early remodeling of the ventricle as early as 2 weeks after banding since apoptosis was seen at 4 weeks in previous publication. This study addressed only the changes of hERG potassium channel mRNA expression. It has been known that the expression of other ion channels could also have been changed especially in the model of cardiac failure. Hence the future study should also address the changes of other cardiac ion channels (i.e., Ito, IK1, IKs, etc.). This may help to elucidate the shortening of the epicardial MAP90 and improve understand the effects that could be observed when evaluating torsadogenic and non-torsadogenic drugs in this rabbit model. Furthermore, IKr current must be obtained from epicardium and endocardium of both ventricles in order to confirm the gain function of hERG channel in the rabbit model of RVH. Acknowledgment This study was supported by the 90th Anniversary of Chulalongkorn University Fund (Ratchadaphiseksomphot Endowment Fund), QTest Labs, LLC, and Ratchadaphiseksompot Endowment Fund from Chulalongkorn University (Special Task Force for Activating Research (GSTAR 56-008-31-001). The authors would like to thank Dr. Somporn Techangamsuwan for assistance with the histopathological study. References Aidonidis, I., Poyatza, A., Stamatiou, G., Lymberi, M., Stamatoyannis, N., & Molyvdas, P.A. (2009). Does-related shortening of ventricular tachycardia cycle length after administration of the KATP channel opener bimakalim in a 4-day-old chronic anesethetized pig model. Journal of Cardiovascular Pharmacology and Therapeutics, 14, 2222–2230. Antzelevitch, C. (2007). Role of spatial dispersion of repolarization in inherited and acquired sudden cardiac death syndromes. American Journal of Physiology - Heart and Circulatory Physiology, 293, H2024–H2038. Barbhaiya, C., Po, J., & Hanon, S. (2013). Tpeak-tend and Tpeak-tend/QT ratio as markers of ventricular arrhythmia risk in cardiac resynchronization therapy patients. Pacing and Clinical Electrophysiology, 36, 103–108. Benoist, D., Stones, R., Drinkhill, M., Bernus, O., & White, E. (2011). Arrhythmogenic substrate in hearts of rats with monocrotaline-induced pulmonary hypertension and right ventricular hypertrophy. American Journal of Physiology - Heart and Circulatory Physiology, 300, H2230–H2237. Bonnet, P.S., Bonnet, J., Boissiere, J.L., Le Net, M., Gautier, R.E., Dumas de la Roque, E., et al. (2004). Chronic hypoxia induces nonreversible right ventricle dysfunction and dysplasia in rats. American Journal of Physiology - Heart and Circulatory Physiology, 287, H1023–H1028. Braun, M.U., Szalai, P., Strasser, R.H., & Borst, M.M. (2003). Right ventricular hypertrophy and apoptosis after pulmonary artery banding: Regulation of PKC isozymes. Cardiovascular Research, 59, 658–667. Carlsson, L., Abrahamsson, C., Andersson, B., Duker, G., & Schiller-Linhardt, G. (1993). Proarrhythmic effects of the class III agent almokalant: Importance of infusion rate, QT dispersion, and early after depolarizations. Cardiovascular Research, 27, 2186–2193. Cuspidi, C., Sala, C., Muiesan, L.M., Luca, D.N., & Schillac, G. (2013). Right ventricular hypertrophy in systemic hypertension: An updated review of clinical studies. Journal of Hypertension, 31, 1–8. Danik, S., Cabo, C., Chiello, C., Kang, S., Wit, A.L., & Coromilas, J. (2002). Correlation of repolarization of ventricular monophasic action potential with ECG in the murine heart. American Journal of Physiology - Heart and Circulatory Physiology, 283, H372–H381. Franz, M.R. (1999). Current status of monophasic action potential recording: Theories, measurements and interpretations. Cardiovascular Research, 41, 25–40.

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